Method of treatment and agents useful for same

ABSTRACT

The present invention relates generally to a method for the treatment and prophylaxis of inflammatory conditions. The present invention is predicated in part on the identification of cells of the monocyte/macrophage lineage being critical for inflammation and, in particular, chronic inflammation. In accordance with the present invention, it is proposed that the reduction in levels of monocyte/macrophage-type cells and/or a reduction in the production of inflammatory and pro-inflammatory mediators by these cells, especially locally, is effective in reducing inflammatory conditions. The present invention further provides animal models useful for screening for reducing levels of monocyte/macrophage-type cells and/or reducing the production of inflammatory and pro-inflammatory mediators of these cells.

[0001] This application is a continuation-in-part of provisional patentapplication U.S. Ser. No. 60/202,392, filed May 8, 2000, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to a method for thetreatment and prophylaxis of inflammatory conditions. The presentinvention is predicated in part on the identification of cells of themonocyte/macrophage lineage being critical for inflammation and, inparticular, chronic inflammation. In accordance with the presentinvention, it is proposed that the reduction in levels ofmonocyte/macrophage-type cells and/or a reduction in the production ofinflammatory and pro-inflammatory mediators by these cells, especiallylocally, is effective in reducing inflammatory conditions. The presentinvention further provides animal models useful for screening forreducing levels of monocyte/macrophage-type cells and/or reducing theproduction of inflammatory and pro-inflammatory mediators of thesecells.

BACKGROUND OF THE INVENTION

[0003] Cytokines impart signals to cells to regulate the immuneresponse, control cell proliferation and differentiation and to regulatethe operation of the cytokine network. However, the pleiotropic natureof cytokines is such that in addition to their beneficial effects,cytokines can also induce adverse side effects such as alteration ofnormal cell growth, undesirable modulation of the functional activity ofother cytokines or other unwanted immune effects such as severeinflammation, fever, malaise, nausea or modulation of haemopoiesis.

[0004] Inflammation is a particularly important physiological processwith effects ranging from minor discomfort to life threateningsituations. Macrophages are the key cells in chronic inflammatorylesions which produce a range of inflammatory and pro-inflammatorymediators such as Tumour Necrosis Factor α (TNF-α) and Interleukin-1(IL-1). TNF-α and IL-1 are proposed to be associated with rheumatoidarthritis, inflammatory bowel disease, Crohn▭s disease, type I diabetes,multiple sclerosis, psoriasis and chronic obstructive lung disease, suchas asthma, chronic bronchitis, emphysema and chronic obstructive airwaydisease. It has been previously proposed to use anti-inflammatoryglucocorticoids, which act, at least in part, to suppress macrophageproduction of TNF-α, IL-1 and other pro-inflammatory mediators. However,although effective, the use of anti-inflammatory glucocorticoids posestherapeutic difficulties given that their receptors are widespread inthe body.

[0005] In work leading up to the present invention, the inventorsproposed to adopt a strategy of lowering monocyte/macrophage levelsand/or reducing their level of activation, especially at a site ofinflammation in the treatment of inflammatory conditions. The methoddeveloped by the instant inventors does not require knowledge of whichpro-inflammatory mediator(s) is(are) more relevant to a particularinflammatory condition than another pro-inflammatory mediator.

SUMMARY OF THE INVENTION

[0006] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

[0007] One aspect of the present invention contemplates a method forameliorating the effects of inflammation in a subject, said methodcomprising administering an agent which inhibits or otherwiseantagonizes the effects of a colony stimulating factor on cells of themonocyte/macrophage lineage thereby reducing the level of proliferation,activation, growth and/or survival of said cells.

[0008] In a particularly preferred aspect, the present inventionprovides a method for ameliorating the effects of inflammation in asubject, said method comprising administering an agent which inhibits orotherwise antagonizes the effects of two or more colony stimulatingfactors on cells of the monocyte/macrophage lineage thereby reducing thelevel of proliferation, activation, growth and/or survival of saidcells.

[0009] Another aspect of the present invention contemplates a method forameliorating the effects of inflammation in a subject, said methodcomprising administering an agent which inhibits or otherwiseantagonizes the effects on M-CSF and/or GM-CSF on cells of themonocyte/macrophage lineage thereby reducing the level of proliferation,activation, growth and/or survival of said cells.

[0010] Yet another aspect of the present invention provides a method forameliorating the effects of inflammation in a subject, said methodcomprising administering an agent which inhibits or otherwiseantagonizes the activity of u-PA and optionally other inflammatorymediators produced by a cell of the monocyte/macrophage lineage.

[0011] Still yet another aspect of the present invention provides amethod for ameliorating the effects of inflammation in a subject, saidmethod comprising administering one or more agents to antagonize theeffects of a colony-stimulating factor on cells of themonocyte/macrophage lineage thereby reducing cell proliferation,activation, growth and/or survival and which antagonize the effects ofu-PA and optionally other inflammatory mediators produced by said cellsof monocyte/macrophage lineage.

[0012] Even still another aspect of the present invention is directed toa composition comprising one or more molecules capable of antagonizingthe ability of a colony-stimulating factor from activating,proliferating, inducing growth and/or survival of cells of amonocyte/macrophage lineage, said composition further comprising one ormore pharmaceutically acceptable carriers and/or diluents.

[0013] More particularly, the present invention provides a compositioncomprising one or more agents capable of antagonizing the effects of twoor more colony-stimulating factors on cells of the macrophage lineagethereby reducing cell proliferation and/or activation and optionally anagent which antagonizes the effects of u-PA produced by said cells ofmacrophage lineage.

[0014] Even still yet another aspect of the present invention provides acomposition comprising two immunointeractive molecules wherein one isspecific for M-CSF and another is specific for GM-CSF, said compositionfurther comprising one or more pharmaceutically acceptable carriersand/or diluents wherein said composition is useful in the treatment ofinflammation.

[0015] Another aspect of the present invention is directed to acomposition comprising an antagonist of u-PA and optionally anantagonist of one or more other inflammatory mediators produced by cellsfrom monocyte/macrophage lineage and one or more pharmaceuticallyacceptable carriers and/or diluents.

[0016] Yet another embodiment of the present invention provides acomposition comprising immunointeractive molecules to M-CSF and GM-CSFand an antagonist of u-PA and optionally one or more antagonists to oneor more other inflammatory mediators produced by cells of amonocyte/macrophage lineage and one or more pharmaceutically acceptablecarriers and/or diluents.

[0017] Still yet another embodiment of the present inventioncontemplates a method for ameliorating the effects of inflammation in asubject, said method comprising administering to said subject aneffective amount of an agent comprising a monocyte/macrophageinteracting ligand chemically linked to an active portion and whereinsaid agent is in an encapsulated form such that the monocyte/macrophageinteracting portion is represented on the outer surface of saidencapsulation while the active portion is part of the encapsulated wallor is internal relative to the encapsulation wall such that amonocyte/macrophage cell is capable of internalizing said encapsulatedagent and wherein said active portion of said agent antagonizescolony-stimulating factor mediated proliferation, activation, growthand/or survival of said monocyte/macrophage cell and/or antagonizes theproduction or activity of one or more inflammatory mediators from saidmonocyte/macrophage cells.

[0018] Even yet another embodiment of the present invention is directedto a method for ameliorating the effects of inflammation in a subject,said method comprising administering to said subject an effective amountof an agent comprising a monocyte/macrophage interacting ligandchemically linked to an active portion or a pro-active form thereof andwherein said active portion of said agent antagonizes colony-stimulatingfactor mediated proliferation, activation, growth and/or survival ofsaid monocyte/macrophage cell and/or antagonizes the production oractivity of one or more inflammatory mediators from saidmonocyte/macrophage cells.

[0019] Another aspect of the present invention is directed to an agentcomprising a monocyte/macrophage interacting ligand chemically linked toan active portion and wherein said agent is in an encapsulated form suchthat the monocyte/macrophage interacting portion is represented on theouter surface of said encapsulation while the active portion is part ofthe encapsulated wall or is internal relative to the encapsulation wallsuch that a monocyte/macrophage cell is capable of internalizing saidencapsulated agent and wherein said active portion of said agentantagonizes colony-stimulating factor mediation, proliferation,activation, growth and/or survival of said monocyte/macrophage and/orantagonizes the production or activity of one or more inflammatorymediators.

[0020] Yet another aspect of the present invention comprisesadministering to said subject an effective amount of an agent comprisinga monocyte/macrophage interacting ligand chemically linked to an activeportion or a pro-active form thereof and wherein said active portion ofsaid agent antagonizes colony-stimulating factor mediated proliferation,activation, growth and/or survival of said monocyte/macrophage celland/or antagonizes the production or activity of one or moreinflammatory mediators from said monocyte/macrophage cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a diagrammatic representation highlighting thetherapeutic concept behind the present invention. In essence,macrophages are the critical targets in chronic inflammation.Colony-stimulating factors such as M-CSF and GM-CSF stimulate survival,proliferation and activation of macrophages resulting in production ofpro-inflammatory mediators such as IL-1, TNF-α and/or u-PA.

[0022]FIG. 2A is a graphical representation showing collagen-inducedarthritis (CIA) development in GM-CSF-deficient mice and littermatecontrols. The incidence of arthritis (shown as cumulative %) of GM-CSF−/− (n=15), GM-CSF +/− (n=28) and GM-CSF +/+ (n=13) mice is shown withtime following primary immunization with CII. The final incidence ofarthritis was significantly lower in the GM-CSF −/− mice compared to theGM-CSF +/+ and GM-CSF +/− mice (p<0.005 for each, X² test). The terms“−/+”, “+/−” and “+/+” relate to homozygous or heterozygous absence (−)or present (+) of a particular gene or allele.

[0023]FIG. 2B is a graphical representation showing the effect ofanti-GM-CSF antibody on CIA development. DBA/1 mice were given a boostof collagen type II at day 21. At day 27, CIA positive mice (n=10/group)were given 300 μg (i.p. daily for 10 days) of either purified monoclonalanti-GM-CSF IgG or isotype control. Daily mean clinical scores (±SEM)are presented.

[0024]FIG. 2C is a graphical representation showing the effect ofanti-M-CSF antibody on CIA development. Same protocol as for FIG. 1Bexcept that mice were given either purified monoclonal anti-M-CSF IgG orisotype control.

[0025]FIG. 3A is a graphical representation showing that anti-GM-CSFtherapy prevents COPD-like inflammation in mice. Levels of neutrophilsrecovered in lung lavage fluid (BAL) reflect the intensity ofinflammation induced by the bacterial wall component, lipopolysaccharide(LPS) (10 μg administered transnasally). Anti-GM-CSF (22E9) therapydose-dependently suppressed the inflammatory response when administered3 hrs prior to LPS. Note that very high dose glucocorticoid steroidtherapy (dexamethasone) produced only slight suppression. Data areexpressed as means ±SEM for n=10-20 mice/data point. The actual amountsof Ab used were {fraction (1/100)} the quantities shown.

[0026]FIG. 3B is a graphical representation showing theanti-GM-CSF-therapy reverses COPD-like inflammation in the lung.Anti-GM-CSF therapy (300 μg/mouse) prevented LPS-induced neutrophiliaeven when added 3-6 hrs after the stimulus. Data are expressed as means±SEM for n=10-20 mice.

[0027]FIG. 3C is a graphical representation showing the suppression ofmacrophage replication by anti-GM-CSF therapy in experimental COPD inmice. Macrophage replication is quantified by the number of mitoticfigures observed histologically. Data are expressed as means ±SEM forn=10-20 mice.

[0028]FIG. 3D is a graphical representation showing suppression of TNF-αformation by anti-GM-CSF therapy of experimental COPD in mice.Anti-GM-CSF therapy suppressed TNF-α levels in BAL of mice given LPS (10μg transnasally administered). The antibody was given 3 hrs prior to LPSadministration and TNF-α levels determined by ELISA 6 hrs later. Dataare mean values ±SEM (n=10 mice).

[0029]FIG. 4 is a graphical representation showing collagen-inducedarthritis (CIA) development in u-PA −/− deficient mice and wild-typecontrols. The severity of arthritis, expressed as a clinical score ofu-PA −/− (n=50) and wild-type control (n=50) mice is shown with timefollowing primary immunization with CII. There was no detectable diseasein the u-PA −/− mice.

[0030]FIG. 5 is a graphical representation showing that anti-GM-CSFinhibits lipopolysaccharide (LPS)-induced exacerbation of asthma. Balb/cmice were sensitized to ovalbumin and challenged over 3 days by aerosolof qualbumin to elicit eosinophilic lung inflammation. Neutrophilicinflammation was elicited by instilling 100 ng/ml LPS into the lungs andmeasuring responses 24 hrs later. Data are shown as mean lung lavagecell counts/ml fluid ±SEM for n=8-10 mice/group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention is predicated in part on a two tierstrategy for controlling inflammation in a subject. At one level, it isproposed to reduce macrophage proliferation, activation, growth and/orsurvival. At a second level, it is proposed to reduce the level ofproduction of pro-inflammatory mediators such as but not limited toIL-1, TNF-α and/or the neutral proteinase, u-PA. The latter level in thestrategy is particularly amenable to local therapeutic intervention,i.e. at the site of inflammation. The two tier strategy may be adoptedseparately or in combination.

[0032] Accordingly, one aspect of the present invention contemplates amethod for ameliorating the effects of inflammation in a subject, saidmethod comprising administering an agent which inhibits or otherwiseantagonizes the effects of a colony stimulating factor on cells of themonocyte/macrophage lineage thereby reducing the level of proliferation,activation, growth and/or survival of said cells.

[0033] In a particularly preferred aspect, the present inventionprovides a method for ameliorating the effects of inflammation in asubject, said method comprising administering an agent which inhibits orotherwise antagonizes the effects of two or more colony stimulatingfactors on cells of the monocyte/macrophage lineage thereby reducing thelevel of cell proliferation, activation, growth and/or survival of saidcells.

[0034] In a related embodiment, the present invention provides a methodfor ameliorating the effects of inflammation in a subject, said methodcomprising administering an agent which inhibits or otherwiseantagonizes the activity of u-PA and optionally other inflammatorymediators produced by a cell of the monocyte/macrophage lineage.

[0035] The preferred cells of the monocyte/macrophage lineage aremacrophages. However, the present invention extends to any other relatedcells which are activated, proliferate, growth and/or survival inresponse to colony stimulating factors and/or which mediate an effectvia inflammatory mediators (e.g. u-PA).

[0036] The term “inflammatory condition” is used in its broadest sensebut preferably relates to chronic inflammation conditions such asrheumatoid arthritis, inflammatory bowel disease, Crohn▭s disease, typeI diabetes, multiple sclerosis, psoriasis and chronic inflammatory lungdisease such as asthma, chronic bronchitis, emphysema or chronicobstructive airway disease. Generally, but not exclusively, theinflammatory condition is localized. Frequently, inflammation involvespro-inflammatory mediators such as TNF-α and IL-1 amongst othersincluding u-PA.

[0037] The term “ameliorate” is used here in its broadest sense to referto the reduction of the symptoms exhibited in patients suffering from aninflammatory condition. It also covers the more specific response ofreducing the levels of inflammatory and pro-inflammatory mediators suchas macrophage colony-stimulating factor (M-CSF), granulocyte, macrophagecolony-stimulating factor (GM-CSF), IL-1, TNF-α, Interleukin 6 (IL-6),products of COX-2, u-PA amongst other molecules. The term “ameliorate”also covers a method of treating a subject exhibiting inflammation.

[0038] In a preferred embodiment, at least two colony-stimulatingfactors are targeted for antagonism. Most preferably, the twocolony-stimulating factors are M-CSF and GM-CSF or related molecules. Arelated molecule is one which at the functional, physiological,immunological or structural level is similar to M-CSF or GM-CSF.

[0039] Accordingly, another aspect of the present invention contemplatesa method for ameliorating the effects of inflammation in a subject, saidmethod comprising administering an agent which inhibits or otherwiseantagonizes the effects on M-CSF and/or GM-CSF on cells of themonocyte/macrophage lineage thereby reducing the level of cellproliferation, activation, growth and/or survival.

[0040] The term “agent” is used in its broadest sense to include acomposition comprising a single valent molecule capable of inhibiting acolony-stimulating factor such as M-CSF or GM-CSF or a multivalentmolecule capable of inhibiting two or more colony stimulating factors ortwo or more molecules which separately or in combination inhibit two ormore colony-stimulating factors. The molecules making up an agentinclude soluble colony-stimulating factor receptors, binding proteins toa colony-stimulating factor, immunointeractive molecules (e.g.antibodies) capable of interacting with a colony-stimulating factor orits receptor and molecules such as those found through natural productscreening which act as antagonists of colony-stimulating factor-receptorinteraction. When the antagonizing molecules are antibodies, these maybe produced in the same species as the subject being treated or they maybe made in another species but rendered immunologically similar to ahomologue from the species being treated. For example, when the subjectis a human, a mouse anti-human antibody to M-CSF or GM-CSF may beemployed in humans once the antibody has been humanized.

[0041] Natural product screening includes the screening of environmentssuch as plants, coral, sea and river beds and micro-organisms formolecules and generally small molecules, which are capable of acting asantagonists of colony-stimulating factor-receptor interaction.Alternatively, chemical libraries may be screened for syntheticchemicals which act in a similar manner. In yet a further alternative,the receptor for the colony-stimulating factor such as the M-CSF andGM-CSF receptors are produced in soluble form. This acts as anantagonist by competing for the receptor on the monocyte/macrophagelineage cells for the colony-stimulating factors.

[0042] Another useful form of the agent exploits the biology ofmonocyte/macrophage surface proteins and internalization mechanisms. Inthis regard, the preferred agents do not activate themonocyte/macrophages but are internalized by the monocyte/macrophagecells. In one embodiment, an agent comprises amonocyte/macrophage-interacting ligand chemically linked to an activeportion such as but not limited to a drug, antibody or pro-drug. A“drug” includes a nucleic acid molecule such as a sense, antisense orribozyme molecule. A “pro-drug” is a molecule which, upon activationsuch as chemical activation, becomes active.

[0043] Conveniently, but not necessarily, the chemical linkage is in theform of an enzyme sensitive bond. Furthermore, the agent may beencapsulated in a material which permits exposure of themonocyte/macrophage interacting ligand. For example, the agent isformulated in a lipid bilayer or equivalent including an artificialmembrane. In an alternative embodiment, the agent is not encapsulatedand is administered directly.

[0044] Presentation of the formulation to a monocyte/macrophage permitsinternalization and, generally, dissociation of the ligand from theactive portion. The active portion may be any of a number of moleculessuch as those which reduce expression of colony-stimulating factorreceptor formation, which antagonizes the signal induced by acolony-stimulating factor and/or which antagonize the production of oneor more inflammatory mediators such as but not limited to u-PA.

[0045] Conveniently, the monocyte-macrophage interacting ligands aresugars.

[0046] Accordingly, another aspect of the present invention contemplatesa method for ameliorating the effects of inflammation in a subject, saidmethod comprising administering to said subject an effective amount ofan agent comprising a monocyte/macrophage interacting ligand chemicallylinked to an active portion and wherein said agent is in an encapsulatedform such that the monocyte/macrophage interacting portion isrepresented on the outer surface of said encapsulation while the activeportion is part of the encapsulated wall or is internal relative to theencapsulation wall such that a monocyte/macrophage cell is capable ofinternalizing said encapsulated agent and wherein said active portion ofsaid agent antagonizes colony-stimulating factor mediation,proliferation, activation, growth and/or survival of saidmonocyte/macrophage and/or antagonizes the production or activity of oneor more inflammatory mediators.

[0047] Particularly preferred colony-stimulation factors include M-CSFand/or GM-CSF.

[0048] Particularly preferred inflammatory mediators include u-PA.

[0049] In yet another embodiment, the agent is provided in anon-encapsulated form.

[0050] According to this embodiment, there is provided a method forameliorating the effects of inflammation in a subject, said methodcomprising administering to said subject an effective amount of an agentcomprising a monocyte/macrophage interacting ligand chemically linked toan active portion or a pro-active form thereof and wherein said activeportion of said agent antagonizes colony-stimulating factor mediatedproliferation, activation, growth and/or survival of saidmonocyte/macrophage cell and/or antagonizes the production or activityof one or more inflammatory mediators from said monocyte/macrophagecells.

[0051] The present invention further contemplates the agents useful inthe practice of the instant method.

[0052] Accordingly, in one embodiment, the present invention is directedto an agent comprising a monocyte/macrophage interacting ligandchemically linked to an active portion and wherein said agent is in anencapsulated form such that the monocyte/macrophage interacting portionis represented on the outer surface of said encapsulation while theactive portion is part of the encapsulated wall or is internal relativeto the encapsulation wall such that a monocyte/macrophage cell iscapable of internalizing said encapsulated agent and wherein said activeportion of said agent antagonizes colony-stimulating factor mediation,proliferation, activation, growth and/or survival of saidmonocyte/macrophage and/or antagonizes the production or activity of oneor more inflammatory mediators.

[0053] In another embodiment, the agent comprises administering to saidsubject an effective amount of an agent comprising a monocyte/macrophageinteracting ligand chemically linked to an active portion or apro-active form thereof and wherein said active portion of said agentantagonizes colony-stimulating factor mediated proliferation,activation, growth and/or survival of said monocyte/macrophage celland/or antagonizes the production or activity of one or moreinflammatory mediators from said monocyte/macrophage cells.

[0054] Alternative agents which target colony-stimulating factors and/orinflammatory mediators include agents capable of stimulating a local orsystemic immune response to a colony-stimulating factor and/or aninflammatory medication and small molecules based on pharmacore drugtemplate models of colony-stimulator factors, their receptors and/orinflammatory mediators such as u-PA.

[0055] The subject is preferably a human but the instant inventionextends to primates, livestock animals, laboratory test animals,companion animals and captured wild animals.

[0056] With respect to blocking u-PA activity, again, this may beaccomplished using anti-u-PA antibodies, antagonists located in naturalproduct screening or screening of chemical libraries, as discussedabove. Furthermore, in a particularly preferred embodiment, the u-PAantagonist treatment may applied locally to the site of inflammationsuch that the antagonizing effects are maintained at the site ofinflammation. A similar approach may be adopted for other inflammatorymediators.

[0057] Either treatment strategy may be adopted or both strategies maybe adopted in tandem.

[0058] According to the latter embodiment, the present inventionprovides a method for ameliorating the effects of inflammation in asubject, said method comprising administering one or more agents toantagonize the effects of a colony-stimulating factor on cells of themonocyte/macrophage lineage thereby reducing cell proliferation,activation, growth and/or survival and which antagonize the effects ofu-PA and optionally other inflammatory mediators produced by said cellsof monocyte/macrophage lineage.

[0059] A single valent or single multivalent agent may be employed ormultiple agents may be used each containing antagonists for thecolony-stimulating factors and/or the inflammatory mediators such asu-PA.

[0060] Preferably, the colony-stimulating factors are M-CSF and GM-CSF.

[0061] Preferably, the cells of monocyte/macrophage lineage aremacrophages.

[0062] Other agents useful in the practice of the present inventioninclude mimotopes or their chemical or biological equivalents.Antibodies are produced in response to B cell-recognized epitopespresent on macromolecular biological molecules. The epitope can be alinear sequence of amino acids or a discontinuous epitope of chemicalgroups spatially arranged, i.e. a conformational epitope. Therecognition and binding site of an antibody can be mapped using smallpeptides. The resultant peptide is referred to herein as a ▭mimotope▭.This may be made from all L amino acids or a mixture of D and L aminoacids. Amino acids not found in human proteins can also be used to makemimotope. The mimotope mimics the epitopes generating the antibody. Thisis useful as a drug design template or as an antigen to produce amonoclonal antibody to an epitope that is difficult to characterize.Mimotopes recognized by anti-GM-CSF and anti-M-CSF monoclonal antibodiesare encompassed by the present invention. Furthermore, a drug designtemplate is also encompassed by the present invention. In this template,cyclic peptides are made and screened in bioassays to determine theminimal structure and constraints. Based on this information, a templateis designed which, after modification, leads to low molecular weightblockers. Such blockers are useful as therapeutic agents. The presentinvention further contemplates a variety of mimetics such as mimetics ofthe mimotopes.

[0063] The agents of the present invention are preferably in a formsuitable for pharmaceutical use.

[0064] Agents useful in modulating the effects of colony-stimulatingfactors in mediated monocyte/macrophage proliferation, activation,growth and/or survival can be usefully screened and assessed using arange of animal models including animals having reduced levels of one ormore colony-stimulating factors through either genetic manipulationand/or antibody or colony-stimulating factor antagonist therapy.

[0065] The present invention is directed to a number of animal modelsuseful in assessing the efficacy of agents useful in the treatment ofinflammation. In one model, an asthma-like inflammation of the airwaysis elicited in mice or other suitable animal by administration ofovalbumin proteins absorbed to an adjuvant. The ovalbumin may beadministered to the animal by any suitable means such as by injection(e.g. i.p. injection) or via nasal administration (e.g. via aerosol.This stimulates an immune response and causes an eosinophilicinflammation of the lung reminiscent of asthma. The model has predictivevalue for drugs active against asthma and also allergic drugs activeagainst the underlying “TH2 immune deviation” elicited by immunization.

[0066] In another model, the COPD model, a COPD-like neutrophil-richmacrophage-activation dependent inflammation is elicited in animals suchas mice by transnasal instillation of the bacterial cell wall componentlipopolysaccharide (LPS) with or without prior manipulation or treatmentof the animal. The ongoing inflammatory response involves the cytokinesTNF-α and chemokines. A characteristic of this inflammatory response isits resistance to the anti-inflammatory effects of glucocorticosteroids.Animals responded with marked-dose dependent neutrophilic inflammation,release of inflammatory cytokines and macrophage-activation. The modelhas predictive value for human COPD, bronchitis and emphysema.

[0067] Yet another model, the exacerbation model, an animal model isemployed wherein an asthma response is elicited in a manner similar tothe asthma model using three challenges in three consecutive doses.Lipopolysaccharide is also instilled into the lungs of mice to elicit aneutrophilic/macrophage inflammatory response with an underlyingeosinophilic inflammation. The model has predictive value foridentifying treatments of exacerbation of human asthma.

[0068] Still a further model employs the administration of methylatedBSA followed by the subcutaneous administration of a cytokine such asGM-CSF and/or M-CSF. Such animals develop a condition similar toarthritis and are useful for screening for anti-inflammatory drugs (e.g.anti-GM-CSF and/or anti-M-CSF agents).

[0069] All these models are encompassed and covered by the presentinvention as are agents identified for the treatment and inflammationusing such models. Furthermore, the present invention contemplates invitro models. One such model employs cell-based and biochemical assaysas part of a drug screening protocol. Monocyte/macrophage populationsare the preferred cell-based assays. Preferred biochemical assaysinclude protease, kinase and cytokine assays. Such assays screen foragents which modulate monocyte/macrophage protease, kinase or cytokinefunction or which modulate the activity of pro-inflammatory effectormolecules.

[0070] Preferably, the animal models are mice or other rodents and mostpreferably are inbred mice, outbred mice or genetically altered mice orcross-hybrids thereof. The preferred strains are Balb/c, C57/B16, c129and congenic or genetically modified derivatives of these strains, beigemice and mice genetically susceptible to asthma-like or COPD-like asthmaresponses.

[0071] Another aspect of the present invention is directed to acomposition comprising one or more molecules capable of antagonizing theability of a colony-stimulating factor from activating, proliferating,inducing growth and/or survival of cells of a monocyte/macrophagelineage, said composition further comprising one or morepharmaceutically acceptable carriers and/or diluents.

[0072] The composition may comprise two antagonists, one for eachcolony-stimulating factor. Alternatively, a single, multivalentantagonist is employed capable of antagonizing two or morecolony-stimulating factors. Yet a further alternative provides acomposition in a two part format with a single valent agent in eachcomposition.

[0073] In a particularly preferred embodiment, the colony-stimulatingfactors are M-CSF and GM-CSF. Preferably, the antagonists areimmunointeractive molecules such as antibodies.

[0074] Accordingly, the present invention provides a compositioncomprising two immunointeractive molecules wherein one is specific forM-CSF and another is specific for GM-CSF, said composition furthercomprising one or more pharmaceutically acceptable carriers and/ordiluents wherein said composition is useful in the treatment ofinflammation.

[0075] Yet another embodiment of the present invention is directed to acomposition comprising an antagonist of u-PA and optionally anantagonist of one or more other inflammatory mediators produced by cellsfor monocyte/macrophage lineage and one or more pharmaceuticallyacceptable carriers and/or diluents.

[0076] Still yet another embodiment of the present invention provides acomposition comprising immunointeractive molecules to M-CSF and GM-CSFand an antagonist of u-PA and optionally one or more antagonists to oneor more other inflammatory mediators produced by cells of amonocyte/macrophage lineage and one or more pharmaceutically acceptablecarriers and/or diluents.

[0077] For convenience, the terms “agent”, “composition” and“pharmaceutical composition” are used herein interchangeably.

[0078] Administration of the agent, such as in the form of apharmaceutical composition, may be performed by any convenient means.The agent is contemplated to exhibit therapeutic activity whenadministered in an amount which depends on the particular case. Thevariation depends, for example, on the human or animal and the agentchosen. A broad range of doses may be applicable. Considering a patient,for example, from about 0.1 μg to about 1 mg of agent may beadministered per dosing, and more preferably 0.1 μg to about 50 μg perdosing. Dosage regimes may be adjusted to provide the optimumtherapeutic response. For example, doses may be administered daily,weekly, monthly or other suitable time intervals or the dose may beproportionally reduced as indicated by the exigencies of the situation.The agent may be administered in a convenient manner such as by theoral, intravenous (where water soluble), intranasal, intraperitoneal,intramuscular, intracranial, intraocular, intrathecal, intracerebrally,infusion, subcutaneous, intradermal or suppository routes or implanting(e.g. using slow release molecules) as well as via direct lung delivery.They may also be administered rectally or using a vapour or aerosol.

[0079] Compositions suitable for injectable use include sterile aqueoussolutions (where water soluble) and/or emulsions or liposomepreparations or sterile powders for the extemporaneous preparation ofsterile injectable solutions. They must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of micro-organisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, gylcerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The preventions of the action of micro-organisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmersal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin or encapsulation in a biocompatible polymersuch as PLA polylactic and/or PLG polyglcolic acid.

[0080] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby sterilization. Dispersions are also contemplated and these may beprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, apreferred method of preparation includes vacuum drying and thefreeze-drying technique which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution.

[0081] When the active ingredients are suitably protected they may beorally administered, for example, with an inert diluent or with anassimilable edible carrier, or it may be enclosed in hard or soft shellgelatin capsule, or it may be compressed into tablets. For oraltherapeutic administration, the active compound may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage 5will be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 2000 mg of active compound.

[0082] The tablets, troches, pills, capsules and the like may alsocontain the components as listed hereafter. A binder such as gum,acacia, corn starch or gelatin; excipients such as dicalcium phosphate;a disintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

[0083] The present invention also extends to forms suitable for topicalapplications such as creams, lotions and gels. This is particularlyadvantageous for the treatment of local inflammation. Furthermore, formssuitable for inhalable administration such as aerosols, powders andinstillations are encompassed by the present invention. The term▭inhalable administration▭ includes direct lung delivery.

[0084] Pharmaceutically acceptable carriers and/or diluents include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, use thereof in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

[0085] It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activematerial and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activematerial.

[0086] Effective amounts of the composition contemplated by the presentinvention will vary depending on the severity of the pain and health andage of the recipient. In general, effective amounts may vary from 0.01ng/kg/body weight to about 1 mg/kg/body weight and preferably 0.01 ng/kgbody weight to about 1 μg/kg body weight. Alternative amounts range from0.1 ng/kg/body weight is about 10 μg/kg/body weight or from 1.0 ng/kgbody weight to about 80 μg/kg body weight.

[0087] The pharmaceutical composition may also comprise geneticmolecules such as a vector capable of transfecting target cells wherethe vector carries a nucleic acid molecule or derivative or analoguethereof capable of expressing a cytokine or cytokine receptor. Thevector may, for example, be a viral vector.

[0088] The agent may also be linked to a targeting means such asmonoclonal antibody, which provides specific delivery of the agent to atarget region.

[0089] In accordance with a method of the invention, the immunogenicagent may be co-administered with one or more other compounds ormolecules. For example, the agent may be co-administered with anothermolecule designed to reduce or alleviate any one or more symptoms of aninflammatory response. By “co-administered” is meant simultaneousadministration in the same formulation or in two different formulationsvia the same or different routs or sequential administration by the sameor different routes. By “sequential administration” is meant a timedifference of from seconds, minutes, hours or days between theadministration of the two types of molecules. These molecules may beadministered in any order.

[0090] As stated above, the present invention extends to the use ofgenetic means to reduce colony-stimulating factor-induced activation andproliferation of cells of macrophage lineage. For example, a geneticconstruct comprising a sequence of nucleotides which, for example, actas antisense molecules to colony-stimulating factor receptorgene-specified mRNA. Alternatively, the nucleotide sequence defines aribozyme specific for the target mRNA. Yet another alternative providethe colony-stimulating factor receptor gene in sense orientation toinduce co-suppression. A similar approach may be used to down-regulateu-PA. The genetic construct may be administered as part of a viralvector or may be administered as a naked nucleic acid molecule.Administration is conveniently but not exclusively via injection.

[0091] The present invention further contemplates, in a preferredembodiment, the use of a M-CSF antagonist and a GM-CSF antagonist in themanufacture of a medicament for the treatment of inflammation in asubject.

[0092] In an alternative embodiment, the present invention provides forthe use of a u-PA antagonist and/or an antagonist of one or more otherpro-inflammatory mediators for the manufacture of a medicament for thetreatment of inflammation in a subject.

[0093] In yet a further embodiment, the present invention is directed tothe use of a M-CSF antagonist, a GM-CSF antagonist and a u-PA antagonistand/or antagonist of one or more other pro-inflammatory mediators in themanufacture of a medicament for the treatment of inflammation in asubject.

[0094] The term “antagonist” is intended to be considered in itsbroadest sense to include molecules which inhibit the activity of thecolony-stimulating factor such as M-CSF and GM-CSF or the activity ofu-PA or other inflammatory mediators. The term also includes geneticmolecules which are useful in down-regulating expression or translationof genes encoding the receptors for the colony-stimulating factors orreducing levels of u-PA.

[0095] As stated above, the composition may also be in the form of anencapsulated or non-encapsulated agent.

[0096] The present invention further extends to the specific targetingof GM-CSF in the treatment of neutrophil-mediated inflammatory. It isproposed that GM-CSF promotes neutrophil survival and activation.Accordingly, anti-GM-CSF therapy is proposed to be useful in thetreatment of neutrophil-mediated inflammation (see FIG. 3A).

[0097] The present invention is further described by the followingnon-limiting Examples.

EXAMPLE 1 Therapeutic Concept

[0098] The therapeutic concept upon which the present invention is basedis shown FIG. 1 In essence, targeting colony-stimulating factors such asM-CSF and GM-CSF results in fewer and/or less activemonocytes/macrophages available to produce inflammatory cytokines andother mediators such as u-PA.

EXAMPLE 2 M-CSF and GM-CSF in Inflammatory Diseases

[0099] Murine arthritis models and murine lung models are used to testthe concept outlined in Example 1. From the arthritis models, it hasbeen found that M-CSF and GM-CSF exacerbate disease. From both geneknockout and antibody blocking data, direct evidence has now beenobtained by the inventors for their involvement in disease progression.In FIG. 2, it can be seen that GM-CSF −/− mice develop minorcollagen-induced arthritis (CIA) disease in the occasional digit.Surprisingly, it was found that the humoral response (four isotypes) tocollagen type II (CII) was not compromised. The results are shown inFIG. 2A.

[0100] A monoclonal antibody to GM-CSF prevents the onset of arthritisand suppresses established disease (FIG. 2B). The anti-GM-CSF mAbblocking in the CIA model provides more efficacy than the alleviationachieved by anti-TNF-α therapy in the same model. Similar results areobserved using anti-M-CSF mAb blocking antibody (FIG. 2C).

EXAMPLE 3 M-CSF and GM-CSF in Inflammatory Lung Disease

[0101] In both a murine COPD model, as well as in a murine allergic(asthma) inflammation model, blocking mAbs to both CSFs suppressesdisease. FIG. 3A shows that blocking antibody to GM-CSF can preventendotoxin-induced lung neutrophilia (a COPD model) even when added afterthe stimulus (FIG. 3B). This provides support for the use of anti-GM-CSFtherapy in the treatment of neutrophil-mediated inflammation in additionto macrophage-mediated inflammation and/or tissue damage transformation.FIG. 3C demonstrates that this antibody suppresses local macrophageproliferation in the same model. FIG. 3D demonstrates the suppression ofTNF-a formation by anti-GM-CSF therapy. The favourable effect ofanti-GM-CSF therapy compared to that found with a glucocorticoid isworth noting (FIG. 3A). The latter class of drugs have potentanti-inflammatory activity but COPD patients are notably refractory tothem. The data in FIG. 3A are consistent with this weak clinicalefficacy; the data are also consistent with the observation thatglucocorticoids do not inhibit the production of GM-CSF by ex vivo lungtissue.

EXAMPLE 4 u-PA/plasmin in Inflammatory Disease

[0102] The inventors developed u-PA −/− mice. FIG. 4 supports theconcept that u-PA is one way in which M-CSF and GM-CSF behave aspro-inflammatory cytokines. u-PA −/− mice do not develop CIA (FIG. 4).These data are similar to what has ben found for this model in GM-CSF−/− (FIG. 2A) and M-CSF −/− mice.

EXAMPLE 5 Animal Models

[0103] 1. Asthma

[0104] Asthma-like inflammation of the airways is elicited in mice byi.p. injection of ovalbumin protein (grade V, 10 μg/animal) absorbed tothe adjuvant Al(OH)₃ on day 0 and day 14. Commencing on day 21, animalsreceive a challenge with ovalbumin solution delivered eithertransnasally (50 μl volume) while under anaesthetic or by aerosol whileconscious. This restimulates the immune system and causes aneosinophilic inflammation of the lung reminiscent of asthma. If thechallenge is repeated, e.g. once daily for three days or intermittentlyover weeks, pathophysiological features of asthma observed in the acutemodels can be elicited more strongly, e.g. bronchial hyper-reactivity,mucus induction and airway wall remodelling. The model has predictivevalue for drugs active against asthma and also anti-allergic drugsactive against the underlying ▭TH2 immune deviations elicited by theimmunization and for identifying mechanisms of disease. Key cytokinesknown to affect the system include but are not limited to IL-4, IL-5,TNF-α, IFN-γ, IL-12, IL-13, IL-19, IL-10 and IL-1.

[0105] 2. COPD

[0106] A COPD-like neutrophil-rich and macrophage activation-dependentinflammation is elicited in mice by instillation, under anaesthetic, ofthe bacterial cell well component, lipopolysaccharide (LPS) dose,usually 1 pg-100 ng/mouse. The ongoing acute inflammatory responseinvolves the cytokines TNF-α and chemokines. A characteristic of thisinflammatory response is its resistance to the anti-inflammatory effectsof glucocorticosteroids. Animals respond with marked dose-dependentneutrophilic inflammation, release of inflammatory cytokines andmacrophage activation. After several days, macrophages typicallyproliferate, a feature also seen in human COPD. The model has predictivevalue for identifying mechanisms and treatments of human COPD,bronchitis and emphysema.

[0107] 3. Novel exacerbation model

[0108] Sudden neutrophilic inflammation of the airways occurs in mostexacerbations of asthma. The inventors have developed a new model wherean asthma response is elicited as described in (1) above using threechallenges on three consecutive days. On day 24, LPS is instilled intothe lungs of mice, usually at 10 ng/mouse, to elicit aneutrophilic/macrophage inflammatory response on top of the underlyingeosinophilic inflammation. The model has predictive value foridentifying mechanisms and treatments of exacerbation of human asthma.

[0109] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features.

1. A method for ameliorating the effects of inflammation in a subject,said method comprising administering an agent which inhibits orotherwise antagonizes the effects of a colony-stimulating factor oncells of the monocyte/macrophage lineage thereby reducing the level ofproliferation, activation, growth and/or survival of said cells.
 2. Amethod according to claim 1 wherein the agent antagonizes the effects oftwo or more colony-stimulating factors.
 3. A method according to claim 1or 2 wherein the colony-stimulating factor is M-CSF, GM-CSF, or M-CSFand GM-CSF.
 4. A method according to claim 1 or 2 further comprising theadministration of an agent which antagonizes u-PA and/or otherinflammatory mediators produced by a cell of the monocyte/macrophagelineage.
 5. A method according to claim 2 wherein the agent is acolony-stimulating factor receptor in soluble form, a binding protein ofa colony-stimulating factor or an antibody to a colony-stimulatingfactor.
 6. A method according to claim 1 or 2 wherein the agent isidentified through natural product screening or screening of a chemicallibrary.
 7. A method according to claim 1 or 2 wherein the agent isinternalized by the monocyte/macrophage.
 8. A method for amelioratingthe effects of inflammation in a subject, said method comprisingadministering one or more agents to antagonize the effects of acolony-stimulating factor on cells of the monocyte/macrophage lineagethereby reducing cell proliferation, activation, growth and/or survivaland which antagonize the effects of u-PA and optionally otherinflammatory mediators produced by said cells of monocyte/macrophagelineage.
 9. A method according to claim 8 wherein the agent antagonizesthe effects of two or more colony-stimulating factors.
 10. A methodaccording to claim 9 wherein the colony-stimulating factor is M-CSF,GM-CSF, or M-CSF and GM-CSF.
 11. A method according to claim 10 whereinthe agent is a colony-stimulating factor receptor in soluble form, abinding protein of a colony-stimulating factor or an antibody to acolony-stimulating factor.
 12. A method according to claim 8 or 9wherein the agent is identified through natural product screening orscreening of a chemical library.
 13. A method according to claim 8 or 9wherein the agent is internalized by the monocyte/macrophage.
 14. Acomposition comprising one or more molecules capable of antagonizing theability of a colony-stimulating factor from activating, proliferating,inducing growth and/or survival of cells of a monocyte/macrophagelineage, said composition further comprising one or morepharmaceutically acceptable carriers and/or diluents.
 15. A compositionaccording to claim 14 wherein the agent antagonizes the effects of twoor more colony-stimulating factors.
 16. A composition according to claim15 wherein the colony-stimulating factor is M-CSF, GM-CSF, or M-CSF andGM-CSF.
 17. A composition according to claim 14 or 15 or 16 furthercomprising an agent which antagonizes the effects of u-PA and/or otherinflammatory mediators produced by cells of a monocyte/macrophagelineage.
 18. A composition comprising an antagonist of u-PA andoptionally an antagonist of one or more other inflammatory mediatorsproduced by cells from monocyte/macrophage lineage and one or morepharmaceutically acceptable carriers and/or diluents.
 19. A compositioncomprising immunointeractive molecules to M-CSF and GM-CSF and anantagonist of u-PA and optionally one or more antagonists to one or moreother inflammatory mediators produced by cells of a monocyte/macrophagelineage and one or more pharmaceutically acceptable carriers and/ordiluents.
 20. A method for ameliorating the effects of inflammation in asubject, said method comprising administering to said subject aneffective amount of an agent comprising a monocyte/macrophageinteracting ligand chemically linked to an active portion and whereinsaid agent is in an encapsulated form such that the monocyte/macrophageinteracting portion is represented on the outer surface of saidencapsulation while the active portion is part of the encapsulated wallor is internal relative to the encapsulation wall such that amonocyte/macrophage cell is capable of internalizing said encapsulatedagent and wherein said active portion of said agent antagonizescolony-stimulating factor mediated proliferation, activation, growthand/or survival of said monocyte/macrophage cell and/or antagonizes theproduction or activity of one or more inflammatory mediators from saidmonocyte/macrophage cells.
 21. A method according to claim 20 whereinthe colony-stimulating factor is M-CSF, GM-CSF, or M-CSF and GM-CSF. 22.A method for ameliorating the effects of inflammation in a subject, saidmethod comprising administering to said subject an effective amount ofan agent comprising a monocyte/macrophage interacting ligand chemicallylinked to an active portion or a pro-active form thereof and whereinsaid active portion of said agent antagonizes colony-stimulating factormediated proliferation, activation, growth and/or survival of saidmonocyte/macrophage cell and/or antagonizes the production or activityof one or more inflammatory mediators from said monocyte/macrophagecells.
 23. A method according to claim 22 wherein the colony-stimulatingfactor is M-CSF, GM-CSF, or M-CSF and GM-CSF.
 24. An agent comprising amonocyte/macrophage interacting ligand chemically linked to an activeportion and wherein said agent is in an encapsulated form such that themonocyte/macrophage interacting portion is represented on the outersurface of said encapsulation while the active portion is part of theencapsulated wall or is internal relative to the encapsulation wall suchthat a monocyte/macrophage cell is capable of internalizing saidencapsulated agent and wherein said active portion of said agentantagonizes colony-stimulating factor mediation, proliferation,activation, growth and/or survival of said monocyte/macrophage and/orantagonizes the production or activity of one or more inflammatorymediators.
 25. A agent according to claim 24 wherein thecolony-stimulating factor is M-CSF, GM-CSF, or M-CSF and GM-CSF.
 26. Amethod comprising administering to said subject an effective amount ofan agent comprising a monocyte/macrophage interacting ligand chemicallylinked to an active portion or a pro-active form thereof and whereinsaid active portion of said agent antagonizes colony-stimulating factormediated proliferation, activation, growth and/or survival of saidmonocyte/macrophage cell and/or antagonizes the production or activityof one or more inflammatory mediators from said monocyte/macrophagecells.
 27. A method according to claim 26 wherein the colony-stimulatingfactor is M-CSF, GM-CSF, or M-CSF and GM-CSF.
 28. A method according toclaim 3 further comprising the administration of an agent whichantagonizes u-PA and/or other inflammatory mediators produced by a cellof the monocyte/macrophage lineage.